In primary cultures of mesencephalic neurons from rat embryos, dopamine uptake and binding sites for dopamine uptake blockers are expressed simultaneously and increase proportionally during development in vitro. In adult rats binding sites for dopamine uptake blockers are unambiguously located in synaptosomal membranes, whereas in primary cultured dopaminergic neurons they occur mainly in the cytosol. Dopamine uptake blockers appear to bind to cytosolic soluble proteins that leak out when neuronal membranes were perforated by streptolysin-0. The absence of membrane- associated, but presence of cytosolic binding sites for dopamine uptake blockers was also observed in striatal synaptosomes prepared from 19-to-20-day-old fetuses and newborn rats. However, six days after birth only membrane-associated binding sites are present, because the binding of dopamine uptake blockers is similar in intact or streptolysin-0-permeabilized synaptosomes. These data suggest that translocation of these binding sites into neuronal membranes may occur during ontogenesis.